Steroidogenic factor 1 (SF-1, or Ad4bp, is a member of the nuclear hormone receptor family that is important for normal development and steroid hormone production. This transcription factor regulates a variety of genes important to the endocrine system and is essential for formation of adrenal glands and gonads. Expression and gene targeting studies indicated that SF-1 acts in adrenal and gonad formation, is regulated in a sexually dimorphic manner, and is important for the function of these tissues after birth. The critical nature of this transcription factor reveals the need to examine mechanism important to its regulation and function. The major goal of this proposal is to identify and characterize transcription factors regulating SF-1 in the gonads. This will ultimately lead to the discovery of new proteins important for gonadal development and function, proper endocrine regulation, and the enhancement of our understanding of factors controlling fertility. Using transient transfection analysis and DNase I hypersensitivity mapping (aim I) and transgenic mouse studies (aim III), we will uncover the elements needed for correct spatial and temporal expression of SF-1. Protein/DNA interactions will be examined using electrophoretic mobility shift assays and in vivo footprinting, leading to identification and cloning of important proteins. In addition, studies are proposed (aim II) to investigate two proteins, USF1 and USF2, that we have shown interact with an important SF-1 promoter SF-1 promoter element (E-box) in the testis, and thus are implicated in its regulation. Immunohistochemistry and in situ hybridization analysis will be used to examine the expression of these proteins, while co-transfection studies will investigate the direct regulation of the SF-1 promote by these transcription factors.